Antibodies against f glycoprotein of hendra and nipah viruses

ABSTRACT

The present invention relates to antibodies or antibody fragments that bind, neutralize, and/or inhibit Hendra or Nipah virus. The invention provides antibodies or antibody fragments that selectively bind to the F glycoprotein of Hendra or Nipah virus, and pharmaceutical compositions including such antibodies and/or fragments. The invention further provides polynucleotides encoding the antibodies and fragments of the invention and host cells transformed therewith. Additionally, the invention discloses prophylactic, therapeutic, and diagnostic methods employing the antibodies, fragments, polynucleotides, and/or compositions of the invention.

FIELD OF THE INVENTION

This invention relates generally to the field of immunology andspecifically to antibodies and antibody fragments that bind to Hendraand Nipah viruses and/or inhibit Hendra and Nipah virus activities.

BACKGROUND OF THE INVENTION

Nipah virus (NiV) and Hendra virus (HeV) are closely relatedparamyxoviruses that comprise the Henipavirus genus (Anonymous 1999 MMWRMorb Mortal Wkly Rep Ward, J. W. ed. 48:335-337; Chew, M. H. et al. 2000J Infect Dis 181:1760-1763; Chua, K. B. et al. 2000 Ann Neurol48:802-805; Eaton, B. T. 2001 Microbes Infect 3:277-278; Goh, K. J. etal. 2000 N Engl J Med 342:1229-1235; Lee, K. E. et al. 1999 Ann Neurol46:428-432; Lim, C. C. et al. 2000 Am J Neuroradiol 21:455-461; Murray,K. et al. 1995 Science 268:94-97). Paramyxoviruses are negative-senseRNA containing enveloped viruses and encompass a variety of importanthuman and animal pathogens, including measles virus, mumps virus, Sendaivirus, Newcastle disease virus, rinderpest virus, canine distempervirus, human parainfluenza viruses, respiratory syncytial virus, andsimian virus 5 (reviewed in Lamb and Parks, 2007, Fields Virology, eds.Knippe & Howley, Lippincott, Williams & Wilkins, pp. 1449-1496).

Like other paramyxoviruses, HeV and NiV possess two majormembrane-anchored glycoproteins in the envelope of the viral particle.One glycoprotein is required for host cell receptor recognition andattachment and is designated as either a hemagglutinin-neuraminidaseprotein (HN), a hemagglutinin protein (H), or in the case ofhenipaviruses, a glycoprotein (G), which has neither hemagglutinationnor neuraminidase activities. The other major glycoprotein is the fusion(F) glycoprotein, which is a trimeric class I fusogenic envelopeglycoprotein containing two heptad repeat (HR) regions and a hydrophobicfusion peptide (Fp). The henipavirus F glycoprotein is synthesized as aprecursor F₀ that undergoes posttranslational cleavage by host cellCathepsin L that occurs within the endosomal compartment, most likelyduring endocytosis and recycling of F to the mature fusiogenic F₁ (alarger carboxy terminal fragment) +F₂ (a smaller amino terminalfragment) subunits that are held together by disulfide bonds throughconserved cystine residues. See Pager, C. T. et al. 2006. Virology 346:251-7; Pager, C. T. et al. 2005. J Virol 79: 12714-20; Meulendyke, K. A.et al. 2005, J Virol, 79: 12643-9; Diederich, S. M. et al. 2005, J BiolChem, 280: 29899-903. In the mature form of F, the Fp's are situated atthe N terminal of F₁ followed by the first HR (HRA) and the second HR(HRB) is located at the C terminus of F₁ preceding its transmembranedomain (reviewed in Lamb and Parks, 2007, Fields Virology, eds. Knippe &Howley, Lippincott, Williams & Wilkins, pp. 1449-1496).

Following attachment to host receptor ephrin (EFN) B2 or B3 via the Gglycoprotein, HeV and NiV infect cells through a pH-independent membranefusion process. This process is still poorly understood and is believedto involve conformational changes in G upon receptor binding that leadsto activation and triggering of F. Lamb, R. A. et al. 2006, Virology,344:30-7; Steffen, D. L. et al. 2012, Viruses, 4:280-308. Upontriggering, F undergoes significant conformational rearrangements thatfacilitate the insertion of the fusion peptide into target membranes,bringing the two HR regions together in the formation of the six-helixbundle structure or trimer-of-hairpins during or immediately followingfusion of virus and cell membranes. The F driven membrane fusion processis thought to involve an irreversible folding from a metastable formfollowed by subsequent discrete conformational changes to a lower energystate. Several molecular details of this F re-folding upon triggeringhave been revealed in the structural solutions of both post- andpre-fusion conformations of respirovirus F. Yin, H. S. et al. 2005, ProcNatl Acad Sci USA, 102(26): 9288-93; Yin, H. S. et al. 2006, Nature439:38-44.

Although currently there are no clinically approved vaccines ortherapeutics against HeV or NiV, a Henipavirus G glycoprotein specificneutralizing monoclonal antibody (mAb) m102.4 was shown to protectAfrican green monkey against HeV from lethal disease when it wasadministered as late as 72 hours post infection. Bossart, K. N. et al.2011, Sci Transl Med, 3:105ra103. Antibodies or antibody fragments, suchas monoclonal antibodies (mAbs) and fragments thereof, can be useful inelucidating the structure of a protein and understanding the functionassociated with various domains as well as providing a potential reagentfor use as prophylaxis and/or therapeutic agents as in the case of theanti G m102.4. To date, there are very few reported anti henipavirus FmAbs and none are produced from recombinant protein. Aguilar, H. C. etal. 2007,J Virol, 81:4520-32; Guillaume, V. H. et al. 2006, J Virol,80:1972-8. These reports provide limited information concerning thespecific properties of the isolated antibodies. The development of aneutralizing anti-F antibodies and antibody fragments could serve asanother potential henipavirus infection therapeutic agent perhaps moreeffectively when combined with m102.4. The anti-F antibodies andantibody fragments could also provide valuable tool to facilitate instructural and functional characterization of F mediated fusion inhenipaviruses.

Therefore, the development of neutralizing or inhibiting antibodies andantibody fragments against NiV and HeV could have important implicationsfor prophylaxis and passive immunotherapy. In addition, thecharacterization of the epitopes of the antibodies and antibodyfragments and the mechanisms of neutralization and inhibition of NiV andHeV infection could provide helpful information for development ofcandidate vaccines and drugs. Finally, such antibodies and antibodyfragments could also be used for diagnosis and as research reagents.

SUMMARY OF THE INVENTION

The present invention relates to antibodies or antibody fragments thatbind, neutralize, and/or inhibit Hendra and/or Nipah virus. Inparticular, the present invention provides an antibody or fragmentthereof that selectively binds a Hendra virus or Nipah virus Fglycoprotein, wherein said antibody comprises: a heavy chain variableregion comprising at least one complementarily-determining region (CDR)having the amino acid sequence selected from the group consisting of SEQID NO: 35, 37 and 39; and a light chain variable region comprising atleast one CDR having the amino acid sequence selected from the groupconsisting of SEQ ID NO: 43, 45 and 47. The invention also provides ahumanized antibody or antibody fragment selectively binding to a Hendravirus or Nipah virus F glycoprotein, wherein said antibody or antibodyfragment comprises a heavy chain variable region having at least 90%sequence identity to SEQ ID NO: 33.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequence of m5B3 and ScFv construct cartoon. (A)Amino acid sequence of V_(H) and V_(L) of m5B3. The CDR regions arelabeled and underlined and the framework regions (FR) are also marked.The V_(H) and V_(L) of m5B3, humanized 5B3 (h5B3) and humanized 5B3.1(h5B3.1) are separated by a flexible linker (e.g. a connector peptide-(G₄5)₃-) and inserted into a promoter enhanced pcDNA vector with ahygromycin selection marker, an Immunoglobulin light chain (Ic) leadersequence (Igk lead) at the construct N terminal, an S peptide tag(Stag), and a hexa histidine tag (His) at the C terminal.

FIG. 2. Binding of murine 5B3 (m5B3), humanized 5B3 (h5B3), andhumanized 5B3.1 (h5B3.1) with F. (A) Binding of m5B3, h5B3, and h5B3.1ScFv with soluble (sF) and full length (FL) F. The ScFv constructs ofm5B3, h5B3, and h5B3.1 as shown in FIG. 1B were transfected into 293Tcells and supernatant was harvested at 48 hr post transfection. A humanScFv from a human ScFv library was used as control (ctrl). Equal amountof supernatant was added with sF protein or FL F expressing cell lysateand precipitated (IP) with Ni²⁺beads or S protein agarose as indicated.(B) Binding of h5B3, and h5B3.1 IgG with FL F. The human anti HeV G mAb,m102.4 which has the same Fc and CL fragment was used as control mAb.Purified mAb, each 2 Lig, were added to FL F expressing cell lysatefollowed by precipitation with protein G Sepharose. In all cases, theprecipitated products were analyzed on SDS PAGE followed by westernblotting and the blots were probed (IB) with appropriate antibodies todetect the bands as indicated. IP: Immunoprecipitate; IB: Immuno blot;H: Heavy chain; L: Light chain.

FIG. 3. Alignment of framework regions (FR) of m5B3 and h5B3 with thatof human ScFv library and VH sequence of h5B3.1. (A) The FR's of V_(H)and V_(L) of m5B3 were aligned with that of human ScFv library andconserved human residues were identified as indicated by vertical arrowsabove the alignment. These conserved residues were then replaced intom5B3 homologous positions to produce FR's of h5B3 as shown at the firstrow of the alignment. (B) Amino acid sequence of h5B3.1. The CDR regionsare labeled and underlined. Highlighted residues in CDR indicate theamino acids that were mutated in h5B3 to generate h5B3.1.

FIG. 4. Diagram of vectors used to produce h5B3.1 IgG1 in pcDNA andcoomassie stain of purified m5B3, h5B3, and h5B3.1 IgG. (A) PCR primerswith Xho I sites flanking the light and heavy chain ORF of pDR12 asshown by arrows were used to amplify the pDR12 h5B3.1 plasmid DNA, thePCR product was then digested and inserted into the Xho I site in thepromoter enhanced pcDNA3.I Hygro(+) as shown. (B) Purified mAbs asindicated, 4 μg each, were analyzed on SDS PAGE followed by coomassieblue staining. Vertical arrows indicate the heavy (H) and light (L)chains of the mAbs. MW: Molecular weight marker.

FIG. 5. Determination of 5B3 chain binding. Supernatant of cellexpressing different S peptide tagged h5B3 and human ScFv VH VL chimerasas indicated were added to untagged F expressing cell lysate andprecipitated with S protein agarose. The precipitated products wereanalyzed on SDS PAGE followed by western blotting and the blots wereprobed (IB) with anti S peptide antibody to detect the ScFv or anti-Fantibody to detect F.

FIG. 6. Binding of NiV and CedPV F chimeras with different anti NiV FmAbs and fusion activities of the chimeras. (A) Cell lysates expressingdifferent S peptide tagged NiV and CedPV F chimeras were precipitatedwith different anti NiV F mAbs or S protein agarose as indicated. Theprecipitated products were analyzed on SDS PAGE followed by westernblotting and the blots were probed with anti S peptide Ab. Top bandright below the 64 kDa marker is F₀ and lower band right below the 51kDa marker is F₁. The schematic diagrams of the chimeras are shown onthe left of the blots where red and blue indicate CedPV and NiV Fregions. 1E11, 12B2, and 5B3 are murine mAbs against the F glycoprotein.(B) Different NiV and CedPV F chimeras were tested for their ability topromote cell fusion in a β-Gal reporter cell fusion assay byco-expressing with NiV G in receptor negative HeLa-USU cells usingpermissive HeLa-ATCC cells as the target population. Assays wereperformed in triplicate, and fusion results were calculated andexpressed as mean rates of (3-Gal activity (change in optical density at570 nm per minute ×1,000). Ni: NiV; Ce: CedPV; Hd: globular head of F;HRB: heptad repeat B of F.

FIG. 7. Mapping of 5B3 epitope by mutagenesis. (A) Precipitation andwestern blot analysis of murine 5B3 defective F mutants. A panel of Speptide tagged NiV F mutants were generated and expressed in 293T cells.The F expressing cell lysates were divided equally and precipitated with5B3, 12B2, and S protein agarose separately. The mAb-F complex was thenadded with protein G Sepharose. The precipitated products were analyzedon SDS PAGE followed by western blotting and the blots were probed withanti S peptide Ab. Top band is F₀ and lower band is F₁. (B) Fusionactivity of 5B3 defective NiV F mutants in a β-Gal reporter cell fusionassay. The mutants of NiV F shown in (A) were tested for their abilityto promote cell fusion by co-expressing with NiV G in receptor negativeHeLa-USU cells using permissive HeLa-ATCC cells as the targetpopulation. The data shown are the mean percentage of WT fusion levelsmeasured for each mutant calculated from three separate experimentsnormalized with total expression as measured by densitometry of westernblot bands. The bars represent the range from multiple experiments. WT:wild type F. (C) Location of 5B3 epitope mapped to NiV F trimerstructure displayed as surface representation. Different shades of graymarked the subunits of the trimeric F. Colored residues mark those thatwere mutated in this study. Residues in orange were tested but had noeffect on 5B3 binding. Residues in pink are both 5B3 and I2B2 defective.Magenta marked residues that are 5B3 defective but had no effect on 12B2binding. Fusion peptide of the subunit where the epitope is colored isin blue. (D) Zoom in image of (C) labeling all residues tested in thisstudy.

FIG. 8. Mechanism of 5B3 inhibition. (A) sF was cleaved by trypsin toproduce mature F₁ +F₂ and protease inhibitor was added to stop thereaction. 2 μg of biotinylated FC2 peptide was added with differentamount of mAbs as indicated. The samples were then heated to 50° C. for15 min to trigger F. The FC2-sF complex was then precipitated withavidin agarose. (B) Assay was carried out as described in (A) with m5B3and h5B3.1, the unbound material from the 5 μg mAb reaction (4th lanefrom left) was collected and precipitated with protein G Sepharose (5thlane from left). (C) Assay was carried out as described in (A) with 2 μgof mAb and increasing temperature as indicated for the heat treatment.The unbound material from all reactions was collected and precipitatedwith protein G Sepharose. In all the above cases, precipitated productswere analyzed on SDS PAGE followed by western blotting and the blotswere probed with anti-F rabbit antibody to detect F.

TABLE A Brief Description of m5B3, h5B3, and h5B3.1 SEQ ID NOs. HeavyChain SEQ ID NOs Light Chain SEQ ID NOs Fab/mab V_(H) FR1 CDR1 FR2 CDR2FR3 CDR3 FR4 V_(L) FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 m5B3  1  2  3  4  5  6 7  8  9 10 11 12 13 14 15 16 h5B3 17 18 19 20 21 22 23 24 25 26 27 2829 30 31 32 h5B3.1 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g., Singleton P andSainsbury D., Dictionary of Microbiology and Molecular Biology 3^(rd)ed., J. Wiley & Sons, Chichester, New York, 2001, and Fields Virology4^(th) ed., Knipe D. M. and Howley P. M. eds, Lippincott Williams &Wilkins, Philadelphia 2001.

As used herein, the term “antibody” refers to an immunoglobulin moleculethat may have the ability to specifically bind to a particular antigen.Antibodies may have different varieties known as isotypes or classes,such as but not be limited to the five basic antibody isotypes known asIgA, IgD, IgE, IgG and IgM. An antibody fragment may comprise a part ofan immunoglobulin molecule or a combination of parts of immunoglobulinmolecules. Antibody fragments may retain antigen binding ability.Antibody fragment may include antigen binding active fragments such asbut not be limited to the well-known active fragments F(ab′, Fab, Fv,Fc, and Fd as well as fushion peptide such as ScFv. Antibodies andantibody fragments are well known to those of ordinary skill in thescience of immunology. Antibodies and antibody fragments are regularlyemployed for both in vitro and in vivo studies and processes.

As used here, the terms “heavy chain” and “light chain” refer to thewell-known immunoglobulin subunits and as part of an antibody and thefragments of the subunits. In their complete forms, the heavy chain isgenerally a longer polypeptide than the light chain. The heavy chain maycomprise one heavy chain variable region (V_(H)) that is important forbinding antigen and the light chain may comprise one light chainvariable region (V_(L)) that is important for binding the antigen.

The Fab fragment (fragment antigen-binding) is a region of an antibodythat binds to antigens. Fab may comprise one constant and one variabledomain of each of the heavy and the light chain. These domains shape theparatope—the antigen-binding site—at the amino terminal end of themonomer. The two variable domains bind the epitope on their specificantigens. F(ab′)₂ refers to an antibody fragment comprising a dimer ofFab. Fab and F(ab′)₂ may be generated by recombinant technology or bycleavage of an antibody or a fragment of antibody. As is known in theart, only a portion of an antibody molecule, the paratope, is involvedin the binding of the antibody to the epitopes of the antigen. The pFc′and Fc regions (fragment crystallizable region), for example, areeffectors of the complement cascade but are not involved in antigenbinding.

As used here, an ScFv (single-chain variable fragment) is a fusionpeptide of the variable regions of the heavy (V_(H)) and light chains(V_(L)) of immunoglobulins, connected with a connector or linkerpeptide. In some embodiments, the connector peptide ranges from abouttwo to about 50 amino acids. In some embodiments, the connector peptideranges from about ten to about 25 amino acids. The ScFv may retain theantigen binding ability of the original immunoglobulin molecule. Here anScFv is considered an antibody fragment.

As used here, the Fd fragment may comprise the heavy chain portion of aFab fragment. The Fd fragment may be produced by enzymatic cleavage orrecombination technologies. In some embodiments, the Fd fragments arethe major determinant of antibody specificity (a single Fd fragment maybe associated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Complementarity determining regions (CDRs) are peptide regions withinthe antigen-binding portion of an antibody. CDRs may directly interactwith the epitope of the antigen and are the main determinant of antibodyspecificity. The framework regions (FRs) are peptide regions in theantigen-binding portion of the antibody that maintain the tertiarystructure of the paratope. In some embodiments, in both the heavy chainvariable region (V_(H)) and the light chain variable region (V_(L)),there are four framework regions (FR1 through FR4) separatedrespectively by three complementarity determining regions (CDR1 throughCDR3). The CDRs, and in particular the CDR3 regions, and moreparticularly the heavy chain CDR3, may be largely responsible forantibody specificity.

As used herein, the terms “Hendra Virus Disease” and “Nipah VirusDisease” refer to diseases caused, directly or indirectly, by infectionwith Hendra or Nipah virus. The broad species tropisms and the abilityto cause fatal disease in both animals and humans have distinguishedHendra virus (HeV) and Nipah virus (NiV) from all other knownparamyxoviruses (Eaton B. T. 2001 Microbes Infect 3:277-278). Theseviruses can be amplified and cause disease in large animals and can betransmitted to humans where infection is manifested as a severerespiratory illness and/or febrile encephalitis.

As used herein with respect to polypeptides, the term “substantiallypure” means that the polypeptides are essentially free of othersubstances with which they may be found in nature or in vivo systems toan extent practical and appropriate for their intended use. Inparticular, the polypeptides are sufficiently pure and are sufficientlyfree from other biological constituents of their host cells so as to beuseful in, for example, generating antibodies, sequencing, or producingpharmaceutical preparations. By techniques well known in the art,substantially pure polypeptides may be produced in light of thepolynucleotide and amino acid sequences disclosed herein. Because asubstantially purified polypeptide of the invention may be admixed witha pharmaceutically acceptable carrier in a pharmaceutical preparation,the polypeptide may comprise only a certain percentage by weight of thepreparation. The polypeptide is nonetheless substantially pure in thatit has been substantially separated from the substances with which itmay be associated in living systems.

As used herein, “sequence identity” is a measure of the identity ofnucleotide sequences or amino acid sequences compared to a referencenucleotide or amino acid sequence. A polypeptide having an amino acidsequence at least, for example, about 95% “sequence identity” to areference an amino acid sequence, e.g., SEQ ID NO: 1, is understood tomean that the amino acid sequence of the polypeptide is identical to thereference sequence except that the amino acid sequence may include up toabout five modifications per each 100 amino acids of the reference aminoacid sequence. In other words, to obtain a peptide having at least about95% sequence identity to a reference amino acid sequence, up to about 5%of the amino acid residues of the reference sequence may be deleted orsubstituted with another amino acid or a number of amino acids up toabout 5% of the total amino acids in the reference sequence may beinserted into the reference sequence. These modifications of thereference sequence may occur at the N-terminus or C-terminus positionsof the reference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among amino acids in thereference sequence or in one or more contiguous groups within thereference sequence.

In general, the sequences are aligned so that the highest order match isobtained. “Sequence identity” per se has an art-recognized meaning andcan be calculated using well known techniques. While there are severalmethods to measure identity between two polynucleotide or polypeptidesequences, the term “identity” is well known to skilled artisans(Carillo (1988) J. Applied Math. 48, 1073). Examples of computer programmethods to determine sequence identity and similarity between twosequences include, but are not limited to, GCG program package (Devereux(1984) Nucleic Acids Research 12, 387), BLASTP, ExPASy, BLASTN, FASTA(Atschul (1990) J. Mol. Biol. 215, 403) and FASTDB. Examples of methodsto determine sequence identity and similarity are discussed in Michaels(2011) Current Protocols in Protein Science, Vol. 1, John Wiley & Sons.

In one embodiment of the present invention, the algorithm used todetermine sequence identity between two or more polypeptides is BLASTP.In another embodiment of the present invention, the algorithm used todetermine sequence identity between two or more polypeptides is FASTDB,which is based upon the algorithm of Brutlag (1990) Comp. App. Biosci.6, 237-245). In a FASTDB sequence alignment, the query and referencesequences are amino sequences. The result of sequence alignment is inpercent sequence identity. In one embodiment, parameters that may beused in a FASTDB alignment of amino acid sequences to calculate percentsequence identity include, but are not limited to: Matrix=PAM,k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization GroupLength=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, WindowSize=500 or the length of the subject amino sequence, whichever isshorter.

If the reference sequence is shorter or longer than the query sequencebecause of N-terminus or C-terminus additions or deletions, but notbecause of internal additions or deletions, a manual correction can bemade, because the FASTDB program does not account for N-terminus andC-terminus truncations or additions of the reference sequence whencalculating percent sequence identity. For query sequences truncated atthe N- or C- termini, relative to the reference sequence, the percentsequence identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminus to the reference sequencethat are not matched/aligned, as a percent of the total bases of thequery sequence. The results of the FASTDB sequence alignment determinematching/alignment. The alignment percentage is then subtracted from thepercent sequence identity, calculated by the above FASTDB program usingthe specified parameters, to arrive at a final percent sequence identityscore. This corrected score can be used for the purposes of determininghow alignments “correspond” to each other, as well as percentagesequence identity. Residues of the reference sequence that extend pastthe N- or C-termini of the query sequence may be considered for thepurposes of manually adjusting the percent sequence identity score. Thatis, residues that are not matched/aligned with the N- or C-termini ofthe comparison sequence may be counted when manually adjusting thepercent sequence identity score or alignment numbering.

For example, a 90 amino acid residue query sequence is aligned with a100 residue reference sequence to determine percent identity. Thedeletion occurs at the N-terminus of the query sequence and therefore,the FASTDB alignment does not show a match/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe reference sequence (number of residues at the N- and C-termini notmatched/total number of residues in the reference sequence) so 10% issubtracted from the percent sequence identity score calculated by theFASTDB program. If the remaining 90 residues were perfectly matched(100% alignment) the final percent sequence identity would be 90% (100%alignment−10% unmatched overhang). In another example, a 90 residuequery sequence is compared with a 100 reference sequence, except thatthe deletions are internal deletions. In this case the percent sequenceidentity calculated by FASTDB is not manually corrected, since there areno residues at the N- or C-termini of the subject sequence that are notmatched/aligned with the query. In still another example, a 110 aminoacid query sequence is aligned with a 100 residue reference sequence todetermine percent sequence identity. The addition in the query occurs atthe N-terminus of the query sequence and therefore, the FASTDB alignmentmay not show a match/alignment of the first 10 residues at theN-terminus. If the remaining 100 amino acid residues of the querysequence have 95% sequence identity to the entire length of thereference sequence, the N-terminal addition of the query would beignored and the percent identity of the query to the reference sequencewould be 95%.

As used here, the term “conservative substitution” denotes thereplacement of an amino acid residue by another biologically similarresidue. Conservative substitution for this purpose may be defined asset out in the tables below. Amino acids can be classified according tophysical properties and contribution to secondary and tertiary proteinstructure. A conservative substitution is recognized in the art as asubstitution of one amino acid for another amino acid that has similarproperties. Exemplary conservative substitutions are set out in below inTable I.

TABLE I Conservative Substitutions Side Chain Characteristic Amino AcidAliphatic Non-polar Gly, Ala, Pro, Iso, Leu, Val Polar-uncharged Cys,Ser, Thr, Met, Asn, Gln Polar-charged Asp, Glu, Lys, Arg Aromatic His,Phe, Trp, Tyr Other Asn, Gln, Asp, Glu

Alternatively, conservative amino acids can be grouped as described inLehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp.71-77, as set forth below in Table II.

TABLE II Conservative Substitutions Side Chain Characteristic Amino AcidNon-polar (hydrophobic) Aliphatic: Ala, Leu, Iso, Val, Pro Aromatic:Phe, Trp Sulfur-containing: Met Borderline: Gly Uncharged-polarHydroxyl: Ser, Thr, Tyr Amides: Asn, Gln Sulfhydryl: Cys Borderline: GlyPositively Charged (Basic): Lys, Arg, His Negatively Charged (Acidic)Asp, Glu

And still other alternative, exemplary conservative substitutions areset out below in Table III.

TABLE III Conservative Substitutions Original Residue ExemplarySubstitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln,His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H)Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe Leu (L) Ile, Val,Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu,Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y)Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala

As used herein with respect to polypeptides and polynucleotides, theterm “isolated” means: (i) amplified in vitro by, for example,polymerase chain reaction (PCR); (ii) recombinantly produced by cloning;(iii) purified, as by cleavage and gel separation; or (iv) synthesizedby, for example, chemical synthesis. An isolated polynucleotide is onewhich is readily manipulable by recombinant DNA techniques well known inthe art. Thus, a nucleotide sequence contained in a vector in which 5′and 3′ restriction sites are known or for which polymerase chainreaction (PCR) primer sequences have been disclosed is consideredisolated but a polynucleotide sequence existing in its native state inits natural host is not. An isolated polypeptide and polynucleotide maybe substantially purified, but need not be. For example, apolynucleotide that is isolated within a cloning or expression vector isnot pure in that it may comprise only a tiny percentage of the materialin the cell in which it resides. Such a polynucleotide is isolated,however, as the term is used herein because it is readily manipulable bystandard techniques known to those of ordinary skill in the art.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribing and 5′ non-translatingsequences involved with initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Especially, such 5′ non-transcribing regulatory sequences willinclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences, as desired.

As used herein, a “vector” may be any of a number of polynucleotidesinto which a desired sequence may be inserted by restriction andligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids and phagemids. A cloning vector is one which isable to replicate in a host cell, and which is further characterized byone or more endonuclease restriction sites at which the vector may becut in a determinable fashion and into which a desired DNA sequence maybe ligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification and selection of cells which have beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., (β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques. In some embodiments, the vectors are capableof autonomous replication and expression of the structural gene productspresent in the DNA segments to which they are operably joined.

Novel Anti-HeV and NiV F Glycoprotein Antibodies or Antibody Fragment

The present invention derives, in part, from the development, isolationand characterization of novel antibodies or antibody fragments thatselectively bind to and inhibit Hendra and Nipah viruses. As describedmore fully below, these antibodies or antibody fragments have been shownto bind the F glycoprotein and to reduce or block the infection ofHendra and Nipah viruses. The paratope of the anti-HeV and NiV Fabfragments associated with the neutralization epitopes on the HeV and NiVglycoprotein F are defined by the amino acid (aa) sequences of theimmunoglobulin heavy and light chain regions described in Table A andSEQ ID NO: 1 through SEQ ID NO: 48. Additional antibodies, antibodyfragments, and related sequences are disclosed by SEQ ID NO: 49-80.

In some embodiments, the present invention provides the full-lengthantibodies or antibody fragments thereof selectively binding to Hendraand Nipah F glycoproteins in isolated form and in pharmaceuticalpreparations. Similarly, as described below, the present inventionprovides isolated polynucleotides, vectors, host cells transformed withthe polynucleotides, and compositions and pharmaceutical preparationsincluding isolated polypeptides, which encode the full-length Hendra andNipah F glycoprotein antibodies and/or antibody fragments. Finally, thepresent invention provides methods, as described more fully below,employing these antibodies and polynuecleotides in the in vitro and invivo diagnosis, prevention and therapy of Hendra Virus Disease or NipahVirus Disease.

The complete amino acid sequences of the antigen-binding Fab portions ofthe Hendra and Nipah monoclonal antibodies m5B3 (murine 5B3), h5B3(humanized 5B3) and h5B3.1 (humanized 5B3.1) as well as the relevantV_(H), V_(L) FR and CDR regions are listed in Table A and disclosedherein. SEQ ID NOs: 1, 17 and 33 disclose the amino acid sequences ofthe Fd fragment of the Hendra and Nipah monoclonal antibodies. The aminoacid sequences of the heavy chain FR1, CDR1, FR2, CDR2, FR3, CDR3 andFR4 regions are disclosed as (FR1, SEQ ID NOs: 2, 18 and 34); (CDR1, SEQID NOs: 3, 19 and 35); (FR2, SEQ ID NOs: 4, 20 and 36); (CDR2, SEQ IDNOs: 5, 21 and 37); (FR3, SEQ ID NOs: 6, 22 and 38); (CDR3, SEQ ID NOs:7, 23 and 39); and (FR4, SEQ ID NOs: 8, 24 and 40). SEQ ID NOs: 9, 25and 41 disclose the amino acid sequences of the light chain variablefragments of the Hendra and Nipah antibodies. The amino acid sequencesof the light chain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 regions aredisclosed as (FR1, SEQ ID NOs: 10, 26 and 42); (CDR1, SEQ ID NOs: 11, 27and 43); (FR2, SEQ ID NOs: 12, 28 and 44); (CDR2, SEQ ID NOs: 13, 29 and45); (FR3, SEQ ID NOs: 14, 30 and 46); (CDR3, SEQ ID NOs: 15, 31 and47); (FR4, SEQ ID NOs: 16, 32 and 48).

It is now established in the art that the non-CDR regions of a mammalianantibody may be replaced with similar regions of conspecific orheterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv, Fd and ScFyfragments of Hendra and Nipah F glycoprotein antibodies; chimericantibodies in which the Fc and/or FR1 and/or FR2 and/or FR3 and/or FR4and/or CDR1 and/or CDR2 and/or CDR3 regions of the Hendra and Nipahantibodies have been replaced by homologous human or non-humansequences; chimeric F(ab′)₂ fragments in which the FR1 and/or FR2 and/orFR3 and/or FR4 and/or CDR1 and/or CDR2 and/or CDR3 regions of the Hendraand Nipah F glycoprotein antibodies have been replaced by homologoushuman or non-human sequences; chimeric Fab fragments in which the FRand/or CDR1 and/or CDR2 and/or CDR3 regions have been replaced byhomologous human or non-human sequences; chimeric Fd fragment antibodiesin which the FR1 and/or FR2 and/or FR3 and/or FR4 and/or CDR1 and/orCDR2 and/or CDR3 regions have been replaced by homologous human ornon-human sequences; and ScFv in which the FR1 and/or FR2 and/or FR3and/or FR4 and/or CDR1 and/or CDR2 and/or CDR3 regions have beenreplaced by homologous human or non-human sequence. Thus, those skilledin the art may alter the Hendra and Nipah antibodies by the constructionof CDR grafted or chimeric antibodies or antibody fragments containingall, or part thereof, of the disclosed heavy and light chain V-regionCDR amino acid sequences (Jones, P. T. et al. 1986 Nature 321:522-525;Verhoeyen, M. et al. 1988 Science 39:1534-1536; and Tempest, P. R. etal. 1991 Biotechnology 9:266-271), without destroying the specificity ofthe antibodies for the F glycoprotein epitope. Such FR or CDR grafted orchimeric antibodies or antibody fragments can be effective in preventionand treatment of Hendra or Nipah virus infection in animals (e.g.,horses) and man.

In some embodiments, the antibodies and/or antibody fragments may beproduced in which some or all of the FR regions of the Hendra and NipahF glycoprotein antibodies have been replaced by other homologous humanFR regions. In addition, the Fc portions may be replaced so as toproduce IgA or IgM as well as IgG antibodies bearing some or all of theCDRs of the Hendra and Nipah F glycoprotein antibodies. Of particularimportance is the inclusion of the Hendra and Nipah F glycoproteinantibody heavy chain CDRs, to a lesser extent, the other CDRs of theHendra and Nipah F glycoprotein antibodies. Such humanized antibodiesand/or antibody fragments have particular utility in that they do notevoke an immune response against the antibody itself.

The current invention discloses an antibody or antibody fragment thereofthat selectively binding a Hendra virus or Nipah virus F glycoprotein,wherein said antibody comprises: a heavy chain variable regioncomprising at least one complementarily-determining region (CDR) havingthe amino acid sequence selected from the group consisting of SEQ ID NO:3, 5, 7, 19, 21, 23, 35, 37 and 39; and a light chain variable regioncomprising at least one CDR having the amino acid sequence selected fromthe group consisting of SEQ ID NO: 11, 13, 15, 27, 29, 31, 43, 45 and47. In particular, the current invention discloses an antibody orantibody fragment thereof that selectively binds a Hendra virus or Nipahvirus F glycoprotein, wherein said antibody comprises: a heavy chainvariable region comprising at least one complementarily-determiningregion (CDR) having the amino acid sequence selected from the groupconsisting of SEQ ID NO: 35, 37 and 39; and a light chain variableregion comprising at least one CDR having the amino acid sequenceselected from the group consisting of SEQ ID NO: 43, 45 and 47.

The current invention discloses an antibody or antibody fragment thatmay comprise a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 35, an antibody or antibody fragment that may comprise aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 37;and/or an antibody or antibody fragment that may comprise a heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO: 39. In addition,the current invention discloses an antibody or antibody fragment thatmay comprise a light chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 43, an antibody or antibody fragment that may comprise alight chain CDR2 comprising the amino acid sequence of SEQ ID NO: 45;and/or an antibody or antibody fragment that may comprise a light chainCDR3 comprising the amino acid sequence of SEQ ID NO: 47.

The current invention discloses an antibody or antibody fragmentcomprising a heavy chain variable region with at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQID NO: 1, 17 or 33. The current invention discloses an antibody orantibody fragment comprising a heavy chain variable region with theamino acid sequence of SEQ ID NO: 1, 17 or 33. In some embodiments, theantibody fragment is an ScFv, Fab, F(ab′)₂, or Fd.

The current invention discloses an antibody or antibody fragmentcomprising a light chain variable region with at least 90% or 99%sequence identity to SEQ ID NO: 19, 25 or 41. The current inventiondiscloses an antibody or antibody fragment comprising a light chainvariable region with the amino acid sequence of SEQ ID NO: 9, 25 or 41.In some embodiments, the antibody fragment is an ScFv, Fab, or F(ab′)₂.

The current invention discloses an antibody or antibody fragmentcomprising a heave chain variable region with the amino acid sequence ofSEQ ID NO: 1, 17 or 33, and a light chain variable region with the aminoacid sequence of SEQ ID NO: 9, 25 or 41.

The current invention discloses an antibody or antibody fragmentselectively binding to Hendra virus or Nipah virus F glycoprotein,wherein said antibody or antibody fragment comprises a heavy chaincomprising one or more amino acid sequences selected from the groupconsisting of: FR1 region comprising SEQ ID NO: 2, 18 or 34, FR2 regioncomprising SEQ ID NO: 4, 20 or 36, FR3 region comprising SEQ ID NO: 6,22 or 38, and FR4 region comprising SEQ ID NO: 8, 24 or 40. In addition,the current invention discloses an antibody or antibody fragmentselectively binding to Hendra virus or Nipah virus F glycoprotein,wherein said antibody or antibody fragment comprises a light chaincomprising one or more amino acid sequences selected from the groupconsisting of: FR1 region comprising SEQ ID NO: 10, 26 or 42, FR2 regioncomprising SEQ ID NO: 12, 28 or 44, FR3 region comprising SEQ ID NO: 14,30 or 46, and FR4 region comprising SEQ ID NO: 16, 32 or 48.

In some embodiments, the current invention discloses an ScFv antibodyfragment comprising a V_(H) and a V_(L) having a sequence selected fromthe group consisting of: SEQ ID NO: 1, 17, 33, 9, 25, and 41. The V_(H)and V_(L) may be connected by a connector peptide with a length of 2-50amino acids. In some embodiments, the ScFv fragment may comprise a V_(H)of SEQ ID NO: 33 and a V_(L) of SEQ ID NO: 41, wherein the connectorpeptide may comprise 10-25 amino acids. In some embodiments, theconnector peptide may comprise SEQ ID NO: 52.

In some embodiments, the invention relates to an antibody or antibodyfragment that selectively binds to Hendra virus or Nipah virus Fglycoprotein, wherein said antibody or antibody fragment comprises aheavy chain comprising one or more amino acid sequences selected fromthe group consisting of: FR1 region comprising SEQ ID NO: 34, FR2 regioncomprising SEQ ID NO: 36, FR3 region comprising SEQ ID NO: 38, and FR4region comprising SEQ ID NO: 40, and a light chain comprising one ormore amino acid sequences selected from the group consisting of: FR1region comprising SEQ ID NO: 42, FR2 region comprising SEQ ID NO: 44,FR3 region comprising SEQ ID NO: 46, and FR4 region comprising SEQ IDNO: 48.

The current invention discloses a humanized antibody or antibodyfragment selectively binding to a Hendra virus or Nipah virus Fglycoprotein, wherein said antibody or antibody fragment comprises aheavy chain variable region having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17or 33. In some embodiments, the antibody or antibody fragment comprisesa heavy chain variable region having the amino acid sequence of SEQ IDNO: 17 or 33. The current invention discloses a humanized antibody orantibody fragment selectively binding to a Hendra virus or Nipah virus Fglycoprotein, wherein said antibody or antibody fragment comprises alight chain variable region having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 25or 41. In some embodiments, the antibody or antibody fragment comprisesa light chain variable region having the amino acid sequence of SEQ IDNO: 25 or 41.

In some embodiments, the current invention discloses an antibodycomprising heavy and/or light chain variable regions with 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to theheavy and/or light chain variable regions of the antibody encoded on theplasmids contained in FreeStyle™ 293 cells deposited as American TypeCulture Collection (ATCC) deposit PTA-120575. In some embodiments, thecurrent invention discloses an antibody comprising heavy and light chainvariable regions with identical sequences to the heavy and light chainvariable regions of the antibody encoded on the plasmids contained inFreeStyle™ 293 cells deposited as American Type Culture Collection(ATCC) deposit PTA-120575.

The current invention discloses a humanized antibody or antibodyfragment selectively binding to a Hendra virus or Nipah virus Fglycoprotein eiptope, wherein said antibody or antibody fragmentinhibits Hendra or Nipah virus infection. The current inventiondiscloses a humanized antibody or antibody fragment selectively bindingto a Hendra virus or Nipah virus F glycoprotein eiptope, wherein saidantibody or antibody fragment disrupts virus host membrane fusion. Thecurrent invention discloses a humanized antibody or antibody fragmentselectively binding to a Hendra virus or Nipah virus F glycoproteineiptope, wherein said antibody or antibody fragment blocks Fglycoprotein folding. In some embodiments, the antibody or antibodyfragment inhibits Hendra or Nipah virus infection by disrupting virushost membrane fusion. In some embodiments, the antibody or antibodyfragment disrupts virus host membrane fusion by blocking F glycoproteinfolding.

It is also possible, in accordance with the present invention, toproduce chimeric antibodies or antibody fragments including non-humansequences. Thus, one may use, for example, murine, ovine, equine, bovineor other mammalian Fc or FR sequences to replace some or all of the Fcor FR regions of the Hendra and Nipah F glycoprotein antibodies. Some ofthe CDRs may be replaced as well. Such chimeric antibodies or antibodyfragments bear non-human immunoglobulin sequences admixed with the CDRsof the human Hendra and Nipah F glycoprotein antibodies. Theseantibodies or antibody fragments may be used, among others, for briefperiods or in immunosuppressed individuals. Hendra and Nipah virusesalso infect animals and such antibodies may be used for brief periods orin immunosuppressed subjects.

For inoculation or prophylactic uses, in some embodiments, theantibodies of the present invention are full-length antibody moleculesincluding the Fc region. Such full-length antibodies may have longerhalf-lives than smaller antibody fragments (e.g., Fab) and are moresuitable for intravenous, intraperitoneal, intramuscular, intracavity,subcutaneous, or transdermal administration.

In some embodiments, Fab fragments and other antibody fragments,including not limited to chimeric Fab fragments, may be used. Fabs offerseveral advantages over F(ab′)₂ and whole immunoglobulin molecules forthis therapeutic modality. First, because Fabs have only one bindingsite for their cognate antigen, the formation of immune complexes isprecluded whereas such complexes can be generated when bivalentF(ab′)₂'s and whole immunoglobulin molecules encounter their targetantigen. This is of some importance because immune complex deposition intissues can produce adverse inflammatory reactions. Second, because Fabfragments lack an Fc region they generally cannot trigger adverseinflammatory reactions that are activated by Fc, such as activation ofthe complement cascade. Third, the tissue penetration of the small Fabmolecule is likely to be much better than that of the larger wholeantibody. Fourth, Fab fragments can be produced easily and inexpensivelyin bacteria, such as E. coli, whereas whole immunoglobulin antibodymolecules require mammalian cells for their production in usefulamounts. The latter entails transfection of immunoglobulin sequencesinto mammalian cells with resultant transformation. Amplification ofthese sequences must then be achieved by rigorous selective proceduresand stable transformants must be identified and maintained. The wholeimmunoglobulin molecules must be produced by stably transformed, highexpression mammalian cells in culture with the attendant problems ofserum-containing culture medium. In contrast, production of Fabs in E.coli eliminates these difficulties and makes it possible to producethese antibody fragments in large fermenters which are less expensivethan cell culture-derived products.

In addition to Fab fragments, smaller antibody fragments andepitope-binding peptides having binding specificity for the epitopesdefined by the Hendra and Nipah antibodies can also be used to bind orinhibit the virus. For example, single chain antibodies can beconstructed according to the method of U.S. Pat. No. 4,946,778, toLadner et al. Single chain antibody fragments (e.g. ScFv) comprise thevariable regions of the light and heavy chains joined by a flexiblelinker moiety. Yet smaller is the antibody fragment known as the singledomain antibody or Fd, which comprises an isolated V_(H) single domain.Techniques for obtaining a single domain antibody with at least some ofthe binding specificity of the full-length antibody from which they arederived are known in the art.

It is possible to determine, without undue experimentation, if analtered or chimeric antibody or antibody fragment has the samespecificity as the Hendra and Nipah antibodies by ascertaining whetherthe former blocks the latter from binding to F glycoprotein. If theantibody or fragment thereof being tested competes with a known Hendraor Nipah antibody as shown by a decrease in binding of the Hendra orNipah antibody, then it is likely that the two antibodies and/orantibody fragments bind to the same, or a closely spaced, epitope. Stillanother way to determine whether an antibody has the specificity ofknown Hendra and Nipah antibodies or antibody fragments is topre-incubate the known Hendra or Nipah antibody with F glycoprotein withwhich it is normally reactive, and then add the antibody or antibodyfragment being tested to determine if the antibody or antibody fragmentbeing tested is inhibited in its ability to bind F glycoprotein. If theantibody or antibody fragment being tested is inhibited then, in alllikelihood, it is likely that it has the same, or a functionallyequivalent, epitope and specificity as the known Hendra and Nipahantibodies or antibody fragments of the invention. Screening of Hendraand Nipah antibodies or antibody fragments also can be carried out byutilizing Hendra or Nipah viruses and determining whether the mAbneutralizes the virus.

By using the antibodies or antibody fragments of the invention, it isnow possible to produce anti-idiotypic antibodies or antibody fragmentswhich can be used to screen other antibodies or antibody fragments toidentify whether the antibody or antibody fragment has the same bindingspecificity as an antibody of the invention. In addition, suchantiidiotypic antibodies or antibody fragments can be used for activeimmunization (Herlyn, D. et al. 1986 Science 232:100-102). Suchanti-idiotypic antibodies or antibody fragments can be produced usingwell-known hybridoma techniques (Kohler, G. and Milstein, C. 1975 Nature256:495-497). An anti-idiotypic antibody or antibody fragment is anantibody or antibody fragment which recognizes unique determinantspresent on the antibody produced by the cell line of interest. Thesedeterminants are located in the hypervariable region of the antibody. Itis this region which binds to a given epitope and, thus, is responsiblefor the specificity of the antibody. An anti-idiotypic antibody can beprepared by immunizing an animal with the antibody or antibody fragmentof interest. The immunized animal will recognize and respond to theidiotypic determinants of the immunizing antibody and produce anantibody to these idiotypic determinants. By using the anti-idiotypicantibodies or antibody fragments of the immunized animal, which arespecific for the antibodies or antibody fragments of the invention, itis possible to identify other clones with the same idiotype as theantibody or antibody fragment of the hybridoma used for immunization.Idiotypic identity between antibodies or antibody fragments of two celllines demonstrates that the two antibodies and/or antibody fragments arethe same with respect to their recognition of the same epitopicdeterminant. Thus, by using anti-idiotypic antibodies or antibodyfragments, it is possible to identify other hybridomas expressingantibodies or antibody fragments having the same epitopic specificity.

It is also possible to use the anti-idiotype technology to produceantibodies or antibody fragments which mimic an epitope. For example, ananti-idiotypic antibody or antibody fragment made to a first antibodywill have a binding domain in the hypervariable region which is theimage of the epitope bound by the first antibody. Thus, theanti-idiotypic antibody can be used for immunization, since theanti-idiotype antibody binding domain effectively acts as an antigen.

In some embodiments, the current invention relates to F glycoproteinantibodies and or antibody fragments comprising heavy chain variableregions and/or light chain variable regions and conservativesubsitutions thereof. The specific sequences of the antibodies orantibody fragments are described above. In one aspect, the substitutionsare conservative in nature; however, the invention embracessubstitutions that are also non-conservative.

It should be understood that the definition of peptides or polypeptidesof the invention is intended to include polypeptides bearingmodifications other than insertion, deletion, or substitution of aminoacid residues. By way of example, the modifications may be covalent innature, and include for example, chemical bonding with polymers, lipids,other organic and inorganic moieties. Such derivatives may be preparedto increase circulating half-life of a polypeptide, or may be designedto improve the targeting capacity of the polypeptide for desired cells,tissues or organs. Similarly, the invention further embraces FGFBP3 orvariants thereof that have been covalently modified to include one ormore water-soluble polymer attachments such as polyethylene glycol,polyoxyethylene glycol or polypropylene glycol.

Polynucleotides Encoding Anti-HeV and NiV F Glycoprotein Antibodies orAntibody Fragments

Given the disclosure herein of the amino acid sequences of the heavychain Fd and light chain variable domains of the Hendra and Nipahantibodies or antibody fragments against the F glycoprotein, one ofordinary skill in the art is now enabled to produce polynucleotideswhich encode this antibody or which encode the various antibodyfragments or chimeric antibodies described above. It is contemplatedthat such polynucleotides will be operably joined to otherpolynucleotides forming a recombinant vector for cloning or forexpression of the antibodies of the invention. The present inventionincludes any recombinant vector containing the coding sequences, or partthereof, whether for prokaryotic or eukaryotic transformation,transfection or gene therapy. Such vectors may be prepared usingconventional molecular biology techniques, known to those with skill inthe art, and would comprise DNA coding sequences for the immunoglobulinV-regions of the Hendra and Nipah antibodies, including framework andCDRs or parts thereof, and a suitable promoter either with (Whittle, N.et al. 1987 Protein Eng 1:499-505 and Burton, D. R. et al. 1994 Science266:1024-1027) or without (Marasco, W. A. et al. 1993 Proc Natl Acad SciUSA 90:7889-7893 and Duan, L. et al. 1994 Proc Natl Acad Sci USA91:5075-5079) a signal sequence for export or secretion. Such vectorsmay be transformed or transfected into prokaryotic (Huse, W. D. et al.1989 Science 246:1275-1281; Ward, S. et al. 1989 Nature 341:544-546;Marks, J. D. et al. 1991 J Mol Biol 222:581-597; and Barbas, C. F. etal. 1991 Proc Nati Acad Sci USA 88:7978-7982) or eukaryotic (Whittle, N.et al. 1987 Protein Eng 1:499-505 and Burton, D. R. et al. 1994 Science266:1024-1027) cells or used for gene therapy (Marasco, W. A. et al.1993 Proc Natl Acad Sci USA 90:7889-7893 and Duan, L. et al. 1994 ProcNatl Acad Sci USA 91:5075-5079) by conventional techniques, known tothose with skill in the art.

The expression vectors of the present invention include regulatorysequences operably joined to a nucleotide sequence encoding one of theantibodies of the invention. As used herein, the term “regulatorysequences” means nucleotide sequences which are necessary for orconducive to the transcription of a nucleotide sequence which encodes adesired polypeptide and/or which are necessary for or conducive to thetranslation of the resulting transcript into the desired polypeptide.Regulatory sequences include, but are not limited to, 5′ sequences suchas operators, promoters and ribosome binding sequences, and 3′ sequencessuch as polyadenylation signals. The vectors of the invention mayoptionally include 5′ leader or signal sequences, 5′ or 3′ sequencesencoding fusion products to aid in protein purification, and variousmarkers which aid in the identification or selection of transformants.The choice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art. The subsequentpurification of the antibodies may be accomplished by any of a varietyof standard means known in the art.

In some embodiments, the vector for screening antibodies or antibodyfragment may be a recombinant DNA molecule containing a nucleotidesequence that codes for and is capable of expressing a fusionpolypeptide containing, in the direction of amino- to carboxy-terminus,(1) a prokaryotic secretion signal domain, (2) a polypeptide of theinvention, and, optionally, (3) a fusion protein domain. The vector mayor may not include DNA regulatory sequences for expressing the fusionpolypeptide, e.g. prokaryotic regulatory sequences. Such vectors can beconstructed by those with skill in the art and have been described bySmith, G. P. et al. (1985, Science 228: 13151317); Clackson, T. et al.(1991, Nature 352:624-628); Kang et al. (1991, Methods: A Companion toMethods in Enzymology, vol. 2, R. A. Lerner and D. R. Burton, ed.Academic Press, NY, pp 111-118); Barbas, C. F. et al. (1991, Proc NatlAcad Sci USA 88:7978-7982); Roberts, B. L. et al. (1992, Proc Natl AcadSci USA 89:2429-2433).

A fusion polypeptide may be useful for purification of the antibodies orantibody fragments of the invention. The fusion domain may, for example,include a poly-His tail which allows for purification on Ni⁺ columns orthe maltose binding protein of the commercially available vector pMAL(New England BioLabs, Beverly, Mass.). In some embodiments, the fusiondomain is a filamentous phage membrane anchor. This domain isparticularly useful for screening phage display libraries of monoclonalantibodies but may be of less utility for the mass production ofantibodies. In some embodiments, the filamentous phage membrane anchoris a domain of the cpIII or cpVIII coat protein capable of associatingwith the matrix of a filamentous phage particle, thereby incorporatingthe fusion polypeptide onto the phage surface, to enable solid phasebinding to specific antigens or epitopes and thereby allow enrichmentand selection of the specific antibodies or fragments encoded by thephagemid vector.

The secretion signal is generally a leader peptide domain of a proteinthat targets the protein to the membrane of the host cell, such as theperiplasmic membrane of Gram-negative bacteria. In some embodiments, thesecretion signal for E. coli is a pelB secretion signal. The leadersequence of the pelB protein has previously been used as a secretionsignal for fusion proteins (Better, M. et al. 1988 Science240:1041-1043; Sastry, L. et al. 1989 Proc Natl Acad Sci USA86:5728-5732; and Mullinax, R. L. et al., 1990 Proc Natl Acad Sci USA87:8095-8099). Amino acid residue sequences for other secretion signalpolypeptide domains from E. coli useful in this invention can be foundin Neidhard, F. C. (ed.), 1987 in Escherichia coli and SalmonellaTyphimurium: Typhimurium Cellular and Molecular Biology, AmericanSociety for Microbiology, Washington, D.C.

To achieve high levels of gene expression in E. coli, it may benecessary to use not only strong promoters to generate large quantitiesof mRNA, but also ribosome binding sites to ensure that the mRNA isefficiently translated. In E. coli, the ribosome binding site includesan initiation codon (AUG) and a sequence 3-9 nucleotides long located3-11 nucleotides upstream from the initiation codon (Shine J. andDalgarno L. 1975 Nature 254:34-38). The sequence, which is called theShine-Dalgarno (SD) sequence, is complementary to the 3′ end of E. coli16S rRNA. Binding of the ribosome to mRNA and the sequence at the 3′ endof the mRNA can be affected by several factors: the degree ofcomplementarity between the SD sequence and 3′ end of the 16S rRNA; thespacing lying between the SD sequence and the AUG; and the nucleotidesequence following the AUG, which affects ribosome binding. The 3′regulatory sequences define at least one termination (stop) codon inframe with and operably joined to the heterologous fusion polypeptide.

In some embodiments with a prokaryotic expression host, the vectorutilized includes a prokaryotic origin of replication or replicon, i.e.,a DNA sequence having the ability to direct autonomous replication andmaintenance of the recombinant DNA molecule extrachromosomally in aprokaryotic host cell, such as a bacterial host cell, transformedtherewith. Such origins of replication are well known in the art. Insome embodiments, the origins of replication are those that areefficient in the host organism. In some embodiments, the host cell is E.coli. In some embodiments, for use of a vector in E. coli, the origin ofreplication is ColEl found in pBR322 and a variety of other commonplasmids. In some embodiments, the origin is a p15A origin ofreplication found on pACYC and its derivatives. The ColEl and p15Areplicons have been extensively utilized in molecular biology, areavailable on a variety of plasmids and are described by Sambrook et al.,1989, in Molecular Cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory Press.

In addition, those embodiments that include a prokaryotic replicon mayalso include a gene whose expression confers a selective advantage, suchas drug resistance, to a bacterial host transformed therewith. Typicalbacterial drug resistance genes are those that confer resistance toampicillin, tetracycline, neomycin/kanamycin or chloramphenicol. Vectorstypically also contain convenient restriction sites for insertion oftranslatable DNA sequences. Exemplary vectors are the plasmids pUC18 andpUC19 and derived vectors such as those commercially available fromsuppliers such as Invitrogen (San Diego, Calif.).

When the antibodies or antibody fragments of the invention include bothheavy chain and light chain sequences, these sequences may be encoded onseparate vectors or, more conveniently, may be expressed by a singlevector. The heavy and light chain may, after translation or aftersecretion, form the heterodimeric structure of natural antibodymolecules. Such a heterodimeric antibody may or may not be stabilized bydisulfide bonds between the heavy and light chains.

A vector for expression of heterodimeric antibodies or antibodyfragments, such as the full-length antibodies of the invention or theScFv, F(ab′)₂, Fab or Fv fragment antibodies of the invention, is arecombinant DNA molecule adapted for receiving and expressingtranslatable first and second DNA sequences. That is, a DNA expressionvector for expressing a heterodimeric antibody provides a system forindependently cloning (inserting) the two translatable DNA sequencesinto two separate cassettes present in the vector, to form two separatecistrons for expressing the first and second polypeptides of aheterodimeric antibody. The DNA expression vector for expressing twocistrons is referred to as a dicistronic expression vector.

In some embodiments, the vector comprises a first cassette that includesupstream and downstream DNA regulatory sequences operably joined via asequence of nucleotides adapted for directional ligation to an insertDNA. The upstream translatable sequence may encode the secretion signalas described above. The cassette includes DNA regulatory sequences forexpressing the first antibody polypeptide that is produced when aninsert translatable DNA sequence (insert DNA) is directionally insertedinto the cassette via the sequence of nucleotides adapted fordirectional ligation.

The dicistronic expression vector also contains a second cassette forexpressing the second antibody polypeptide. The second cassette includesa second translatable DNA sequence that may encode a secretion signal,as described above, operably joined at its 3′ terminus via a sequence ofnucleotides adapted for directional ligation to a downstream DNAsequence of the vector that typically defines at least one stop codon inthe reading frame of the cassette. The second translatable DNA sequenceis operably joined at its 5′ terminus to DNA regulatory sequencesforming the 5′ elements. The second cassette is capable, upon insertionof a translatable DNA sequence (insert DNA), of expressing the secondfusion polypeptide comprising a secretion signal with a polypeptidecoded by the insert DNA.

The antibodies or antibody fragments of the present invention mayadditionally be produced by eukaryotic cells such as CHO cells, human ormouse hybridomas, immortalized B-lymphoblastoid cells, and the like. Inthis case, a vector is constructed in which eukaryotic regulatorysequences are operably joined to the nucleotide sequences encoding theantibody polypeptide or polypeptides. The design and selection of anappropriate eukaryotic vector is within the ability and discretion ofone of ordinary skill in the art. The subsequent purification of theantibodies may be accomplished by any of a variety of standard meansknown in the art.

The antibodies or antibody fragments of the present invention mayfurthermore be produced in plants. In 1989, Hiatt A. et al. 1989 Nature342:76-78 first demonstrated that functional antibodies could beproduced in transgenic plants. Since then, a considerable amount ofeffort has been invested in developing plants for antibody (or“plantibody”) production (for reviews see Giddings, G. et al. 2000 NatBiotechnol 18:1151-1155; Fischer, R. and Emans, N. 2000 Transgenic Res9:279-299). Recombinant antibodies can be targeted to seeds, tubers, orfruits, making administration of antibodies in such plant tissuesadvantageous for immunization programs in developing countries andworldwide.

In another embodiment, the present invention provides host cells, bothprokaryotic and eukaryotic, transformed or transfected with, andtherefore including, the vectors of the present invention.

Diagnostic and Pharmaceutical Anti-HeV and NiV F Glycoprotein AntibodyPreparations

The invention also relates to methods for preparing diagnostic orpharmaceutical compositions comprising the antibodies or antibodyfragments of the invention or polynucleotide sequences encoding theantibodies or antibody fragments of the invention, the pharmaceuticalcompositions being used for immunoprophylaxis or immunotherapy of HendraVirus Disease or Nipah Virus Disease. The pharmaceutical preparationincludes a pharmaceutically acceptable carrier. Such carriers, as usedherein, refers to a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Theterm “physiologically acceptable” refers to a non-toxic material that iscompatible with a biological system such as a cell, cell culture,tissue, or organism. The characteristics of the carrier will depend onthe route of administration. Physiologically and pharmaceuticallyacceptable carriers include diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials which are well known inthe art.

The anti-Hendra and anti-Nipah F glycoprotein antibodies or antibodyfragments of the invention may be labeled by a variety of means for usein diagnostic and/or pharmaceutical applications. There are manydifferent labels and methods of labeling known to those of ordinaryskill in the art. Examples of the types of labels which can be used inthe present invention include enzymes, radioisotopes, fluorescentcompounds, colloidal metals, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for binding to the antibodies or antibodyfragments of the invention, or will be able to ascertain such, usingroutine experimentation. Furthermore, the binding of these labels to theantibodies or antibody fragments of the invention can be done usingstandard techniques common to those of ordinary skill in the art.

Another labeling technique which may result in greater sensitivityconsists of coupling the antibodies to low molecular weight haptens.These haptens can then be specifically altered by means of a secondreaction. For example, it is common to use haptens such as biotin, whichreacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, whichcan react with specific anti-hapten antibodies.

The materials for use in the assay of the invention are ideally suitedfor the preparation of a kit. Such a kit may comprise a carrier meansbeing compartmentalized to receive in close confinement one or morecontainer means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method. For example, one of the container means may comprise anantibody and/or antibody fragment of the invention that is, or can be,detectably labeled. The kit may also have containers containingbuffer(s) and/or a container comprising a reporter-means, such as abiotin-binding protein, such as avidin or streptavidin, bound to areporter molecule, such as an enzymatic or fluorescent label.

In vitro Detection and Diagnostics

The antibodies or antibody fragments of the invention are suited for invitro use, for example, in immunoassays in which they can be utilized inliquid phase or bound to a solid phase carrier. In addition, theantibodies or antibody fragments in these immunoassays can be detectablylabeled in various ways. Examples of types of immunoassays which canutilize the antibodies or antibody fragments of the invention arecompetitive and non-competitive immunoassays in either a direct orindirect format. Examples of such immunoassays are the radioimmunoassay(RIA) and the sandwich (immunometric) assay. Detection of antigens usingthe antibodies or antibody fragments of the invention can be doneutilizing immunoassays which are run in either the forward, reverse, orsimultaneous modes, including immunohistochemical assays onphysiological samples. Those of skill in the art will know, or canreadily discern, other immunoassay formats without undueexperimentation.

The antibodies or antibody fragments of the invention can be bound tomany different carriers and used to detect the presence of Hendra orNipah virus. Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylase, natural andmodified cellulose, polyacrylamide, agarose and magnetite. The nature ofthe carrier can be either soluble or insoluble for purposes of theinvention. Those skilled in the art will know of other suitable carriersfor binding antibodies, or will be able to ascertain such, using routineexperimentation.

For purposes of the invention, Hendra or Nipah virus may be detected bythe antibodies or antibody fragments of the invention when present inbiological fluids and tissues. Any sample containing a detectable amountof Hendra or Nipah virus can be used. A sample can be a liquid such asurine, saliva, cerebrospinal fluid, blood, serum or the like; a solid orsemi-solid such as tissues, feces, or the like; or, alternatively, asolid tissue such as those commonly used in histological diagnosis.

In vivo Detection of Hendra or Nipah Virus

In using the antibodies or antibody fragments of the invention for thein vivo detection of antigen, the detectably labeled antibody orantibody fragment is given in a dose which is diagnostically effective.The term “diagnostically effective” means that the amount of detectablylabeled antibody is administered in sufficient quantity to enabledetection of the site having the Hendra or Nipah virus antigen for whichthe antibodies are specific.

The concentration of detectably labeled antibody or antibody fragmentwhich is administered should be sufficient such that the binding toHendra or Nipah virus is detectable compared to the background. Further,it is desirable that the detectably labeled antibody or antibodyfragment be rapidly cleared from the circulatory system in order to givethe best target-to-background signal ratio.

As a rule, the dosage of detectably labeled antibody or antibodyfragment for in vivo diagnosis will vary depending on such factors asage, sex, and extent of disease of the individual. The dosage ofantibody or antibody fragment can vary from about 0.01 mg/kg to about 50mg/kg, e.g. 0.1 mg/kg to about 20 mg/kg, or about 0.1 mg/kg to about 2mg/kg. Such dosages may vary, for example, depending on whether multipleinjections are given, on the tissue being assayed, and other factorsknown to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting an appropriate radioisotope.The radioisotope chosen must have a type of decay which is detectablefor the given type of instrument. Still another important factor inselecting a radioisotope for in vivo diagnosis is that the half-life ofthe radioisotope be long enough such that it is still detectable at thetime of maximum uptake by the target, but short enough such thatdeleterious radiation with respect to the host is acceptable. Ideally, aradioisotope used for in vivo imaging will lack a particle emission butproduce a large number of photons in the 140-250 keV range, which may bereadily detected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to antibodies orantibody fragments either directly or indirectly by using anintermediate functional group. Intermediate functional groups whichoften are used to bind radioisotopes which exist as metallic ions arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetra-acetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to theantibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁷²As, ⁸⁹Zr and ²⁰¹Tl.

The antibodies or antibody fragments of the invention can also belabeled with a paramagnetic isotope for purposes of in vivo diagnosis,as in magnetic resonance imaging (MRI) or electron spin resonance (ESR).In general, any conventional method for visualizing diagnostic imagingcan be utilized. Usually gamma and positron emitting radioisotopes areused for camera imaging and paramagnetic isotopes for MRI. Elementswhich are particularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn,¹⁶²Dy, ⁵²Cr and ⁵⁶Fe.

The antibodies or antibody fragments of the invention can be used invitro and in vivo to monitor the course of Hendra Virus Disease or NipahVirus Disease therapy. Thus, for example, by measuring the increase ordecrease in the number of cells infected with Hendra or Nipah virus orchanges in the concentration of Hendra or Nipah virus present in thebody or in various body fluids, it would be possible to determinewhether a particular therapeutic regimen aimed at ameliorating HendraVirus Disease or Nipah Virus Disease is effective.

Prophylaxis and Therapy of Hendra Virus Disease and Nipah Virus Disease

The antibodies or antibody fragments can also be used in prophylaxis andas therapy for Hendra Virus Disease and Nipah Virus Disease in bothhumans and other animals. The terms, “prophylaxis” and “therapy” as usedherein in conjunction with the antibodies of the invention denote bothprophylactic as well as therapeutic administration and both passiveimmunization with substantially purified polypeptide products, as wellas gene therapy by transfer of polynucleotide sequences encoding theproduct or part thereof. Thus, the antibodies or antibody fragments canbe administered to high-risk subjects in order to lessen the likelihoodand/or severity of Hendra Virus Disease and Nipah Virus Disease oradministered to subjects already evidencing active Hendra or Nipah virusinfection. In the present invention, ScFv or Fab fragments also bind orneutralize Hendra or Nipah virus and therefore may be used to treatinfections.

As used herein, a “prophylactically effective amount” of the antibodiesor antibody fragments of the invention is a dosage large enough toproduce the desired effect in the protection of individuals againstHendra or Nipah virus infection for a reasonable period of time, such asone to two months or longer following administration. A prophylacticallyeffective amount is not, however, a dosage so large as to cause adverseside effects, such as hyperviscosity syndromes, pulmonary edema,congestive heart failure, and the like. Generally, a prophylacticallyeffective amount may vary with the subject's age, condition, and sex, aswell as the extent of the disease in the subject and can be determinedby one of skill in the art. The dosage of the prophylactically effectiveamount may be adjusted by the individual physician or veterinarian inthe event of any complication. A prophylactically effective amount mayvary from about 0.01 mg/kg to about 50 mg/kg, e.g. from about 0.1 mg/kgto about 20 mg/kg, or from about 0.2 mg/kg to about 2 mg/kg, in one ormore administrations (priming and boosting).

As used herein, a “therapeutically effective amount” of the antibodiesor antibody fragments of the invention is a dosage large enough toproduce the desired effect in which the symptoms of Hendra Virus Diseaseor Nipah Virus Disease are ameliorated or the likelihood of infection isdecreased. A therapeutically effective amount is not, however, a dosageso large as to cause adverse side effects, such as hyperviscositysyndromes, pulmonary edema, congestive heart failure, and the like.Generally, a therapeutically effective amount may vary with thesubject's age, condition, and sex, as well as the extent of the diseasein the subject and can be determined by one of skill in the art. Thedosage of the therapeutically effective amount may be adjusted by theindividual physician or veterinarian in the event of any complication. Atherapeutically effective amount may vary from about 0.01 mg/kg to about50 mg/kg, e.g. from about 0.1 mg/kg to about 20 mg/kg, or from about 0.2mg/kg to about 2 mg/kg, in one or more dose administrations daily, forone or several days. In some embodiments, the administration of theantibody is conducted for 2 to 5 or more consecutive days in order toavoid “rebound” of virus replication from occurring.

The antibodies or antibody fragments of the invention can beadministered by injection or by gradual infusion over time. Theadministration of the antibodies or antibody fragments of the inventionmay, for example, be intravenous, intraperitoneal, intramuscular,intracavity, subcutaneous, or transdermal. Techniques for preparinginjectate or infusate delivery systems containing antibodies are wellknown to those of skill in the art. Generally, such systems shouldutilize components which will not significantly impair the biologicalproperties of the antibodies, such as the paratope binding capacity(see, for example, Remington's Pharmaceutical Sciences, 18th edition,1990, Mack Publishing). Those of skill in the art can readily determinethe various parameters and conditions for producing antibody injectatesor infusates without resort to undue experimentation.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and thelike.

Generation of m5B3 ScFv, h5B3 and h5B3.1

Several soluble forms of NiV or HeV F (sF) were engineered andrecombinant sF constructs were produced. Culture supernatant of stable293T cells expressing the different forms of NiV and HeV sF wascollected and clarified prior to affinity chromatography purificationwith S-protein agarose beads (Novagen Corp). The S agarose purifiedmaterial was applied to HiLoad 16/60 Superdex 200 prep grade gelfiltration column to isolate pure trimer. Balb/cJ mice (JacksonLaboratory) were inoculated with different purified soluble viralantigen as shown in table 1 for 4 times at 30 days intervals. Whenindicated the enzymatic S tag cleaved sF was used for immunization. Eachmouse was bled prior to immunization to obtain serum (pre-bleed) asnegative control. In a single immunization, each animal was given 12 μgof sF mixed with Sigma Adjuvant System ™ (Sigma). Each immunization wasgiven in a 0.1 ml dose administered through intraperitoneal andsubcutaneous injections of 0.05 ml in each of 2 sites with a 25 ga.needle. Adjuvant and antigen formulations were made based onmanufacturer's instructions. The mice were bled 7-10 days post 3rdimmunization and serum samples were harvested. Four days beforesacrificing the mice for the final bleed collection, anotherimmunization was performed without adjuvant. All sF glycoproteinconstructs elicit a strong antibody response among immunized mice. TheELISA endpoint titer of each mouse was greater than 1:320,000 in allcases and were able to precipitate native full length F expressed inHeLa cells. In most cases, the harvested serum from the immunized miceinhibits NiV and HeV virus infections.

One form of sF was produced by deleting the transmembrane (TM) andcytoplasmic tail (CT) domains and appending a a trimeric coiled-coil(GCNt) domain. The GCNt-appended constructs (sF_(GCNt)) elicitedcross-reactive henipavirus-neutralizing antibody in mice. In addition,sF_(GCNt) constructs could be triggered in vitro by protease cleavagefollowed by heat treatment. A series of monoclonal antibodies (mAbs)were derived from mice that had been immunized with different sF's, e.g.the non-GCNt-appended NiV sF_(dFp) and NiV sF_(GCNt).

Lymphocytes from immunized mice were fused with the commerciallyavailable Sp2/0 cell line (murine myeloma cells) using high molecularweight polyethylene glycol (PEG) and hybridoma cells were selectedaccording to standard practices. Lymphocytes fused with murine myelomacells were selected for by passage of the cultures in medium withhypoxanthine, aminopterin, and thymadine supplement (HAT, Invirtogen).Hybridoma cell lines secreting antibody reactive with the viral antigenwere identified by enzyme-linked immunosorbent assay (ELISA) usingsupernatant harvested from each well. Colonies secreting mAb which bindssF were isolated and subjected to limiting dilution at least two timesto ensure clonality. Purified mAb was prepared from hybridoma cellsgrown to high density in SFM4MAb medium (Hyclone) supplemented withhypoxanthine and thymadine (HT, Invitrogen) and 100 U/ ml recombinantmouse interleukin 6 (rl L-6, Roche Applied Biosciences). Antibody in thesupernatant of spent cultures was purified using a Protein-G sepharose(GE Healthsciences) bead affinity chromatography. The concentration ofeach preparation was determined using Bradford assay.

Based on ELISA screening of the culture supernatant from the fusionplate, more than 60 hybridoma clones secreting antibodies reacting withsF were identified. 24 clones were selected for further purification vialimiting dilution to generate stable hybridoma lines for mAb isolationand 19 of these mAbs were used for characterization. 18 mAbs are crossreactive for NiV and HeV F and 1 is NiV specificAmong the mAb library,13 were able to precipitate full length F and sF_(GCNt) (pre-fusion F)and the remaining one precipitates only sF_(dEp) (post-fusion F). Of thethirteen prefusion specific mAbs, 12 precipitated F₀ and F₁ and 1 mAbprecipitated only F₀. This observation suggests a different conformationmay be acquired by cleaved and un-cleaved F. Six of the mAbs thatprecipitated only post-fusion F were tested in western blot and found torecognize linear epitopes. Ten mAbs were tested in NiV and HeVpseudotyped virus entry inhibition, showed to inhibit entry and anotheradditional two inhibit at higher concentration. One of the mAb (murine5B3 or m5B3) was tested in live NiV and HeV infection and showed toneutralize at a concentration of 12.5 μg/ml for HeV and 1.5 μg/ml forNiV.

m5B3 was one of the F-specific mAbs. In addition, m5B3 was determined torecognize a conformation-dependent epitope and could also completelyneutralize infectious 200TCID₅₀ NiV and HeV at concentrations of 1.5 and12.5 μg/ml, respectively. Using an immunoprecipitation followed byWestern blot analysis, m5B3 was determined to bind only to NiV and HeVsF_(GCNt) and the full-length wild-type NiV and HeV F-glycoprotein formsof both sF and native F that evidently exist in the pre-fusionconformational state.

The cDNA of 5B3 mouse hybridoma clone was synthesized and the variableregions of heavy (V_(H)) and light chain (V_(L)) sequences wereamplified using several sets of universal primers, cloned, and sequenced(FIG. 1A). The peptide and fragment sequences in FIG. 1A are shown bySEQ ID NO: 1-16, as listed in Table A. A ScFv of murine 5B3 (m5B3), SEQID NO: 49, was then constructed with V_(H) (SEQ ID NO: 1) and V_(L) (SEQID NO: 9) connected by a connector peptide of (G₄S)₃, SEQ ID NO: 52,followed by S peptide and His tag in a promoter modified commerciallyavailable mammalian expression vector pcDNA 3.1 Hygro (+) (InvirogenCorp) (7) (FIG. 1B). Soluble ScFv of m5B3 was expressed in 293FreeStyle™ suspension cell and purified using S agarose affinity columnfollowed by size exclusion chromatography. FIG. 2A shows transientexpressed m5B3 ScFv was able to bind to soluble (sF) and full length(FL) F.

Based on the conserved FR sequence of m5B3, a human ScFv library wasconstructed. The clones from this library were selected based on highlevel of expression in E. coli. The FR sequences of the selected humanScFv clones were aligned with that of m5B3 ScFv. The conserved humanresidues were identified and mutated in the m5B3 FR homologous positionsproducing a humanized 5B3 (h5B3) as shown in FIG. 3A. FIG. 3A, upperpanel, SEQ ID NO: 53—combined V_(H) FR regions of h5B3; SEQ ID NO:54—combined V_(H) FR regions of m5B3; SEQ ID NO: 55-66—human ScFvlibrary clones containing combined V_(H) FR regions as shown in FIG. 3A.FIG. 3A, lower panel, SEQ ID NO: 67—combined V_(L) FR regions of h5B3;SEQ ID NO: 68—combined V_(L) FR regions of m5B3; SEQ ID NO: 69-80—humanScFv library clones containing combined V_(L) FR regions as shown inFIG. 3A.

The h5B3 ScFv was synthesized, cloned, expressed and purified the sameway as m5B3 ScFv. The peptide and fragment sequences of h5B3 are shownby SEQ ID NO: 17-32, as listed in Table A. The h5B3 ScFv has a peptidesequence of SEQ ID NO: 50, wherein the V_(H) (SEQ ID NO: 17) and V_(L)(SEQ ID NO: 25) of h5B3 are connected by a connector peptide (SEQ ID NO:52). Later another version of h5B3 was generated, which was named h5B3.1where one residue on each of the complementarity-determining region(CDR) CDR1 and 2, and two residues on CDR3 were mutated into conservedhuman residues based on the sequence from the human ScFv library asmentioned above (FIG. 3B). The h5B3.1 was then expressed and purified ash5B3 ScFv. The peptide and fragment sequences of h5B3.1 are shown by SEQID NO: 33-48, as listed in Table A. The h5B3.1 ScFv has a peptidesequence of SEQ ID NO: 51, wherein the V_(H) (SEQ ID NO: 33) and V_(L)(SEQ ID NO: 41) of h5B3.1 are connected by a connector peptide (SEQ IDNO: 52).

As shown in FIG. 2A, both h5B3 and h5B3.1 were able to bind FL F.

Generation of h5B3 and h5B3.1 IgG1

The V_(H) and V_(L) of both h5B3 and h5B3.1 were cloned into vectorpDR12 to generate full IgG1. Both h5B3- and h5B3.1-IgG1 were then shownto bind to FL F (FIG. 2B). The open reading frames (ORF) of the heavyand light chain of h5B3.1 IgG1 were cloned into a promoter enhancedexpression vector as shown in FIG. 4A and the construct was used todevelop a stable 293 FreeStyle™ suspension cell line that produced highyield (approximately 8 mg/shaker flask) of h5B3.1 IgG1 in shaker flasksin serum free medium (FIG. 4B).

Freestyle™ cell line 293 cells that contain a plasmid encoding theh5B3.1 antibody were deposited as American Type Culture Collection(ATCC) deposit PTA-120575.

The affinities of m5B3, h5B3- and h5B3.1-IgG1 were then determined asshown in Table 1, which demonstrate the binding kinetics of m5B3, h5B3,and h5B3.1 against sF.

TABLE 1 Binding kinetic analysis of m5B3, h5B3, and h5B3.1 againstsoluble F Rmax Concentration mAb ka (l/Ms) kd (l/s) (RU) of sF KA (l/M)KD (M) χ² m5B3 3.4 × 10⁴ 9.2 × 10⁻⁵ 36.1 0-200 nM 3.7 × 10⁸ 2.7 × 10⁻⁹0.04 h5B3 2.5 × 10⁴ 3.3 × 10⁻⁴ 22.9 0-379 nM 7.5 × 10⁷ 1.3 × 10⁻⁸ 0.03h5B3.1 2.7 × 10⁴ 1.7 × 10⁻³ 24.1 0-279 nM 1.6 × 10⁷ 6.2 × 10⁻⁸ 0.04

Table 1 shows the binding kinetic analysis of m5B3, h5B3, and h5B3.1against soluble F. Biacore analysis was performed by A&G PrecisionAntibody™ (Columbia, Md.) using Biacore 3000. Certificate grade CM5chips were coated with capture antibody (goat anti-mouse IgG Fc formouse mAb and goat anti-human IgG Fc for humananized mAb). The test mAbwas then captured on the chip and the binding kinetics were measured at5 different sF concentrations (from 0 to saturating). Binding kineticparameters at each sF concentration were measured and a χ2 analysis wasperformed to assess the accuracy of the data.

In addition, both h5B3- and h5B3.1-IgG1 were shown to inhibit live NiVand HeV at similar titers as compared to m5B3 (Table 2).

TABLE 2 Virus neutralization by m5B3, h5B3, and h5B3.1 Titer (μg/ml)Titer (μg/ml) 200 TCID50 100 TCID50 mAb NiV HeV NiV HeV m5B3 1.56 12.50.78 1.56 h5B3 3.125 12.5 1.56 0.78 h5B3.1 >100 >100 >100 >100

Table 2 shows virus neutralization by m5B3, h5B3, and h5B3.1. PurifiedmAb were serial diluted in duplicate by doubling dilution starting at100 μg/ml and incubated with NiV or HeV separately at 37° C. for 30 min.The virus-mAb mixture was then used to infect 2×10⁴ Vero cells per wellof a 96 well tissue culture plate. Viral cytopathic effect (cpe) wasobserved at 3 days post infection. The titer was determined as thehighest dilution in which viral cpe was still fully inhibited (absent)in two wells. The assay was performed separately to compare between m5B3and h5B3; and m5B3 and h5B3.1.

Characterization of 5B3 binding and mapping of 5B3 epitope

To determine if both heavy (H) and light (L) chain of 5B3 is involved inbinding to F, chimeras of V_(H) and V_(L) of h5B3 and one of the clonefrom the human ScFv library (FIG. 3A) were constructed as in FIG. 1B. Asshown in FIG. 5, none of the chimeras were able to bind FL F indicatingthat both H and L of 5B3 are involved in binding to F.

Next, to determine the location of 5B3 epitope on F, chimeras of NiV Fand the F protein of Cedar virus (CedPV), a newly discovered henipavirus(Marsh, G. A. et al. 2012, PLoS Pathog, 8:e1002836 in which 5B3 does notreact with were generated. Only the construct that possesses theglobular head domain of NiV F and HRB helical stem, TM and CT of CedPV Fwas able to bind to 5B3 (FIG. 6) indicating that the epitope is locatedat the globular head domain. The head and HRB chimeras were also shownto be functional in a cell-cell fusion assay.

A NiV F mutant, L53D was shown to be defective in binding to 5B3. Basedon the solved crystal structure of sF, residues surrounding L53 weremutated to alanine for hydrophilic and/or hydrophobic residues and/orserine for hydrophobic residues (Table 3) from a WT F construct that hasa C-terminal S peptide tag.

TABLE 3 Summary of F mutants reactivity with 5B3 and 12B2. Binding tomAb Residue Change to 5B3 12B2 N51 A ++ ++ L53 D −− ++ S −− ++ P52 A ++++ T54 A ++ ++ K55 A −− ++ E −− ++ D56 A −− −− E166 A ++ ++ K167 A ++ ++R244 and A and S ++ ++ T245 L246 A ++ ++ D −− −− S +/− +/− G247 A ++ ++S ++ ++ L246 and D and A −− −− G247 G247 and S and S ++ ++ Y248 Y248 A++ ++ S ++ ++ Y248 and D and A −− ++ A249 A249 D +/− ++ S ++ ++ T250 A−− ++ E251 A ++ ++ Y281 A ++ ++ F282 A + ++ S ++ ++ P283 A ++ ++ S + +F282 and A and A −− −− P283 I284 S ++ ++ E251 and A and S −− ++ I284

Table 3 provides a summary of F mutants' reactivities with 5B3 and 12B2,another conformational dependent neutralizing mAb. Residues surroundingL53 as shown in FIG. 7C were mutated to alanine for hydrophilic residuesand/or serine for hydrophobic residues. Single and double F mutants werethen tested for their binding with 5B3 by precipitating the F expressingcell lysate separately with 5B3 and 12B2 by protein G Sepharose and Sprotein agarose. The precipitated products were analyzed on SDS PAGEfollowed by western blotting. The F bands were detected using HRPconjugated anti S peptide antibody. ++: strong binding; +: less binding;+/−: weak binding; −−: no binding.

The F mutants were then tested for their binding with 5B3 byprecipitating F expressing cell lysates with 5B3 and anotherconformational dependent neutralizing mAb, 12B2 to determine if the Fmutant is conformational intact. Equal amount of F expressing celllysate was precipitated with S protein agarose to monitor totalexpression. A summary of the result is shown in table 3 and western blotof selected mutants shown in FIG. 7A. The experiment identified severalsingle F mutants (L53D/S, K55A/E, T250A) and two double mutants(YA248-249AD and E1251-284AS) that are defective in binding to 5B3 buthas no effect on 12B2 binding indicating the location of 5B3 epitope isat the side of the globular head (FIG. 7). Although mutant Y248A alonehad no effect on 5B3 binding, when combined with mutant A249D that isalmost defective (faint band when precipitated by 5B3), the doublemutant is completely defective in 5B3 binding showing that both residuesare important in the binding site. Similarly, mutants E251A and 1284Sare only defective when combined. Mutant F282A showed a slight decreasein 5B3 binding, although completely defective when combined with P283A,this double mutant is also defective in 12B2 binding. Several other 5B3defective mutants (D56A, L246D, and LG246-247DA) were also defective in12B2 binding (FIG. 7A, Table 3). Therefore, these mutants together withthose that are only 5B3 defective were tested in a fusion assay to testfor their ability in promoting cell fusion. As mentioned above mutantL53D is less efficient in promoting fusion, combining mutants L246D andG247A also showed less than 50% of fusion activity compared to wild type(WT) indicating a compromise in function. Only mutant FP282-283AA wascompletely defective in fusion indicating that this double mutant is notconformational and functional intact. Mutants D56A and L246D probablyhave an indirect effect on 12B2 binding that has no effect on F functionin promoting fusion. All other tested mutants retains more than 80% offusion activity except E1251-284AS having less than 40% activity ascompared to WT although maintaining its binding with 12B2 (FIG. 7B).Residues E251 and I284 probably are involved in F protein function inpromoting fusion and perhaps mutation on these residues affect theprotein functionally rather than conformationally as seen by mutantL53D.

Taken together, the location of 5B3 epitope is at the side of theglobular head in a region involving residues L53, K55, Y248, A249, T250,E251, 1284, F282 and possibly residues D56, and L246 (FIG. 7C and D).

Mechanism of 5B3 inhibition

The mechanism of 5B3 inhibition in F fusion was also investigated. An invitro sF triggering assay was previously developed (Chan, Y. P. et al.2012, J Virol. 86: 11457-71) where sF can be triggered by trypsindigestion to its mature F₁+F₂ form followed by heat treatment that willtrigger sF to re-fold into its post fusion conformation. A biotinylatedpeptide with the HRB sequence of F can be added to the trypsin digestedF₁+F₂ that will bind to the intermediate form of the triggering F duringheat treatment and the intermediate form can then be precipitated byavidin agarose. When 5B3 was added together with FC2 peptide, the mAbcan compete with FC2 to bind to the triggering sF in a dose dependentmanner (FIG. 8A). The unbound material from this assay was thenprecipitated with protein G and FIG. 8B shows that both m5B3 and h5B3.1were still bound to sF. Since 5B3 only binds to pre-fusion F, thisindicates that when 5B3 was present during triggering, the mAbstabilizes and held sF in its pre-fusion form. To investigate thisfurther, the triggering was then conducted with varying increasingtemperatures in order to provide more energy to force triggering in thepresence of 5B3. As shown in FIG. 8C, increasing temperatures were ableto recover FC2 precipitation of the intermediate form, indicating thatbinding of 5B3 stabilizes F by creating a higher energy barrier fortriggering to occur.

The results here showed that 5B3 binds to the side of the globular headof F revealing a novel epitope that could be a region important forstabilizing F. One of the residue shown here to be important for 5B3binding, L53 was also shown to be an important residue for the formationof hexameric trimers of F on virus surface which is required forefficient fusion where triggering of a single trimeric F could produce achain effect on the hexamer. Binding of 5B3 to F may also interfere withthis chain triggering effect rendering viral fusion inefficient.

Potential Uses of the Antibodies and Antibody Fragments Against FGlycoprotein

The immediate application of the 5B3 is as a tool for Henipavirusresearch. For example, no mAbs are currently available to detect anddifferentiate pre and post fusion F. 5B3 recognizes aconformation-dependent epitope and can be used to distinguish pre andpost fusion F. These assays are useful in studying the functional andstructural characteristics of the glycoprotein following manipulation,such as conformational changes occurring in F following G receptorbinding. The need for additional diagnostic and detection material forHenipaviruses has arisen from routine epidemics occurring at greaterdistances from the original disease epicenters. Given that Henipavirusesexhibit a broad species tropism of these viruses and the zoonotic originof the viruses often involving domesticated animals in the transmissionto humans, diagnostic and detection techniques should be robust andsuitable for use with samples from many species. Early detection of theHenipaviruses could provide sufficient notice to institute controlmeasures capable of reducing morbidity and mortality. Initial diagnosisof disease emanating from infection with a Henipavirus is dependent onclinical illness and epidemiologic characteristics. The diagnosis islater confirmed by the identification of the virus at a referencelaboratory. 5B3 could be used in the development of a cheap and rapiddiagnostic/detection tool that would be of sufficient specificity andsensitivity to provide early warning to the presence of a Henipavirus.

One of the most promising uses h5B3 and h5B3.1 is in the development ofadditional therapeutic agents for treating Henipavirus infection. Thesehumanized mAb can be expressed, purified in large scale, and used as anantiviral agent without the risk of complications from idiotypicresponses in the recipient. The data from live virus SNT indicated that5B3 is a potent inhibitor of NiV and HeV F mediated fusion. Developmentof h5B3 and/or h5B3.1 as therapeutic agents in combination with the antiG human m102.4 mAb as a cocktail therapy would help minimize thedevelopment of viral resistance to the therapy by simultaneouslytargeting independent epitopes of the two glycoproteins.

The Henipaviruses are some of the most pathogenic and highly fatalemerging viral diseases that have been recognized. Significant advanceshave been made in the study and control of these viruses, but additionalresearch is ongoing. A humanized antivirus mAb has been developed totarget the Henipavirus F glycoprotein that have broad applicability inresearch as well as clinical application. As seen, 5B3 target aconformation-dependent pre fusion specific epitope. Mapping of the 5B3binding region revealed a novel epitope that is important forstabilizing F. The humanized h5B3.1 maintained all binding activitieswith F as well as virus neutralizing titer as compared to m5B3. Theoptimized expression system of h5B3.1 IgG1 developed here provided easy,fast and high yield production of the humanized mAb. Further developmentof h5B3.1 is required and on-going, for example mutagenesis on the CDRregions to improve its binding affinity with F as well as testing itspotency in protecting disease in animal models, but the potentialbenefits and commercial uses are clearly evident.

1. An antibody or fragment thereof that selectively binds a Hendra virusor Nipah virus F glycoprotein, wherein said antibody comprises: a heavychain variable region comprising at least onecomplementarily-determining region (CDR) having the amino acid sequenceselected from the group consisting of SEQ ID NO: 35, 37 and 39; and alight chain variable region comprising at least one CDR having the aminoacid sequence selected from the group consisting of SEQ ID NO: 43, 45and
 47. 2. The antibody or antibody fragment of claim 1 wherein saidantibody or antibody fragment comprises an Fd fragment.
 3. The antibodyor antibody fragment of claim 1 wherein said antibody fragment is a Fabfragment.
 4. The antibody or antibody fragment of claim 1 wherein saidantibody fragment is a single chain variable fragment (ScFv).
 5. Theantibody or antibody fragment of claim 1 wherein said antibody orantibody fragment comprises a heavy chain CDR comprising the amino acidsequence of SEQ ID NO:
 35. 6. The antibody or antibody fragment of claim1 wherein said antibody or antibody fragment comprises a heavy chain CDRcomprising the amino acid sequence of SEQ ID NO:
 37. 7. The antibody orantibody fragment of claim 1 wherein said antibody or antibody fragmentcomprises a heavy chain CDR comprising the amino acid sequence of SEQ IDNO:
 39. 8. The antibody or antibody fragment of claim 1 wherein saidantibody or antibody fragment comprises a heavy chain CDR comprising theamino acid sequence of SEQ ID NO: 35, a heavy chain CDR comprising theamino acid sequence of SEQ ID NO: 37, and a heavy chain CDR comprisingthe amino acid sequence of SEQ ID NO:
 39. 9. The antibody or antibodyfragment of claim 1 wherein said antibody or antibody fragment comprisesa heavy chain comprising a peptide with at least 90% sequence identityto SEQ ID NO:
 33. 10. The antibody or antibody fragment of claim 9wherein said antibody or antibody fragment comprises a heavy chaincomprising a peptide with at least 99% sequence identity to SEQ ID NO:33.
 11. The antibody or antibody fragment of claim 10 wherein saidantibody or antibody fragment comprises a heavy chain amino acidsequence of SEQ ID NO:
 33. 12. The antibody or antibody fragment ofclaim 1 wherein said or antibody fragment antibody comprises a lightchain comprising a peptide with at least 90% sequence identity to SEQ IDNO:
 41. 13. The antibody or antibody fragment of claim 12 wherein saidantibody or antibody fragment comprises a light chain comprising apeptide with at least 99% sequence identity to SEQ ID NO:
 41. 14. Theantibody or antibody fragment of claim 13 wherein said antibody orantibody fragment comprises a light chain amino acid sequence of SEQ IDNO:
 41. 15. The antibody fragment of claim 4 wherein the ScFv furthercomprises a connector peptide.
 16. The antibody fragment of claim 15wherein the connector peptide comprises SEQ ID NO:
 52. 17. The antibodyfragment of claim 4 wherein the ScFv comprises SEQ ID NO:
 51. 18. Ahumanized antibody or antibody fragment selectively binding to a Hendravirus or Nipah virus F glycoprotein, wherein said antibody or antibodyfragment comprises a heavy chain variable region having at least 90%sequence identity to SEQ ID NO:
 33. 19. The humanized antibody orantibody fragment of claim 18, wherein the heavy chain variable regionhas at least 95% sequence identity to SEQ ID NO:
 33. 20. The humanizedantibody or antibody fragment of claim 18, wherein the heavy chainvariable region has at least 99% sequence identity to SEQ ID NO:
 33. 21.The humanized antibody or antibody fragment of claim 18, wherein theheavy chain variable region comprises the amino acid sequence of SEQ IDNO:
 33. 22. The humanized antibody or antibody fragment of claim 18,wherein the antibody is an IgG1.
 23. The humanized antibody or antibodyfragment of claim 18, wherein said antibody or antibody fragmentinhibits Hendra or Nipah virus infection.
 24. The humanized antibody orantibody fragment of claim 23, wherein said antibody or antibodyfragment inhibits Hendra or Nipah virus infection by disrupting virushost membrane fusion.
 25. The humanized antibody or antibody fragment ofclaim 24, wherein said antibody or antibody fragment disrupts virus hostmembrane fusion by blocking F glycoprotein re-folding.
 26. An antibodycomprising heavy and light chain variable regions identical in sequenceto the antibody encoded on the plasmids contained in the FreeStyle™ 293cells deposited as American Type Culture Collection (ATCC) depositPTA-120575.
 27. The antibody of claim 26, wherein the antibody inhibitsHendra or Nipah virus infection by blocking F glycoprotein foldingduring virus host membrane fusion.
 28. A polynucleotide comprising anucleotide sequence encoding the antibody or antibody fragment ofclaim
 1. 29. The polynucleotide of claim 28 wherein said polynucleotideis comprised within a vector.
 30. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and the antibody orantibody fragment of claim
 1. 31. A method for the treatment of HendraVirus Disease or Nipah Virus Disease comprising administering to apatient a therapeutically effective amount of the pharmaceuticalpreparation of claim 30.